Nu-acetonyl-2, 4-dinitropyrrole



United States Patent C) 3,244,726 N-ACETONYL-Z,4-DINITROPYRROLE George Karmas, Bound Brook, NJ., assignor to Urtho Pharmaceutical Corporation, a corporation of New Jersey 7 No Drawing. Original application Nov. 5, 1964, Ser. No. 409,277. Divided and this application Sept. 7, 1965,

Ser. No. 485,551

1 Claim. (Cl. 260-3265) OzNT L lNO:

in which R is a substituent selected from the group consisting of alkyl groups selected from the group consisting of n-propyl, n-butyl, iso-butyl, sec-butyl, n-amyl, isopentyl, sec-pentyl and n-hexyl, haloalkyl groups selected from the group consisting of y-bromopropyl and fi-bromobutyl, acetonyl, B,'y-dihydroxypropyl, fl,' -diacetoxypropyl, l-(N-morpholino carbonyl methyl), piperazino groups selected from the group consisting of l-(N -methyl-N piperazino carbonyl methyl and l-(N -acetyl-N -piperazino carbonyl methyl, and 1-(Z-methyl-S-nitroimidazolyll-ethyl).

Examples of 2,4-dinitropyrroles coming within the scope of the above formula are: l-(y-bromopropyl)-2,4-dinitro pyrrole, 1-acetonyl-2,4-dinitropyrrole, l-isopentyl-2,4-dinitropyrrole, 1 (tiv-dihydroxypropyl)-2,4-dinitropyrrole and l-(N-morpholino carbonyl methyl)-2,4-dinitropyrrole.

The compounds of the present invention have antimicrobial activity and more specifically are effective against Trichomonas foetus, a parasitic protozoan that infects the uterus of animals and causes abortion in cattle. The high order of trichomonodicidal activity possessed by the compounds of the present invention is quite surprising as the closely related 1-alkyl-2,S-dinitropyrroles and 1- alkyl-3,4-dinitropyrroles have no useful activity.

It is an object of the present invention to provide new compounds having therapeutic utility in the treatment of animals infected With Trichomonas foetus.

It is also an object of this invention to provide a new method for the preparation of 1-substituted 2,4-dinitropyrroles.

Heretofore it has been proposed to prepare N-alkylpyrroles by reacting pyrrole with potassium metal in an organic solvent such as a low boiling hydrocarbon and reacting the resulting N-potassiumpyrrole with an alkyl halide for example, methyl iodide. The latter reaction is carried out either by heating the ingredients together in a sealed tube at 120l30 C., or by refluxing in a low boiling organic solvent. By this method, N-methylpyrrole can be obtained in yields around -50%. A considerable amount of pyrrole is converted to u-methylpyrrole, which side reaction is primarily responsible for the relatively low yield of the desired N-alkyl product.

It has also been proposed to react an alkali metal pyrrole in anhydrous liquid ammonia with an organic halide. Under such conditions, high yields have been obtained, but the anhydrous liquid ammonia solvent is more diflicult to handle than a solvent having a lower vapor pressure. Moreover, the alkylation reaction, at

liquid ammonia temperature, requires several hours for completion.

In accordance with the present invention, the alkylation of 2,4-dinitr0pyrroles is effected in dimethylformamide which is a superior solvent for this reaction. In general, the alkali metal salt of 2,4-dinitropyrrole and an excess of the alkylating agent, which may be a chloride, bromide, iodide sulfate, sulfonate, etc., are heated in dimethylformamide for varying periods of time (determined by the reactivity of the alkyla-ting agent). The reaction product is isolated and purified by conventional procedures of extraction, distillation, recrystallization, etc. In those instances where the grouping introduced at the l-position of the pyrrole contains a functional group, further reactions may be performed on this group. For example, an ester may be hydrolyzed to a carboxylic acid and the latter may further be converted to an acid chloride, from which a wide variety of derivatives may be prepared.

The following examples will serve to illustrate more fully the method of preparing the novel compounds of the present invention.

EXAMPLE I 1-butyl-2,4-dinitr0pyrr0le A mixture of 5 grams (0.0275 mole) of the sodium salt of 2,4-dinitropytrrole, 8 milliliters of dibutyl sulfate, and 20 milliliters of dimethylforma-mide is heated to a gentle boil and after one minute is cooled and poured with stirring into a mixture of 200 milliliters of water, grams ice, and 15 grams of sodium carbonate layered with 150 milliliters of other. This mixture is shaken vigorously and then the layers are separated. The ether solution is washed with 100 milliliters of 5% aqueous sodium carbonate, dried with anhydrous magnesium sulfate, concentrated and then distilled. The l-butyl-2,4-dinitropyrrole is a pale yellow oil which distills at l30135 C. at 0.2 mm.

Unalkylated 2,4-dinitropyrrole may be recovered froni the aqueous sodium carbonate solutions by acidification and extraction with ether.

Dimethyl sulfate, diethyl sulfate, and dipropyl sulfate have also been reacted with the sodium salt of 2,4-dinitropyrrole following this procedure.

EXAMPLE II I-isoamyl-2,4-dinitr0pyrr0le A mixture of 5 grams (0.0275 mole) of the sodium salt of 2,4-dinitropyrrole, 6 milliliters of isoamyl bromide, and 10 milliliters of dimethylformamide is heated under reflux for two hours. The reaction mixture is Worked up as described for the l-butyl analog (Example I), and distillation affords 1-isoamyl-2,4-dinitropyrrole as a pale yellow oil which distills at l40 C. at 0.1 mm.

EXAMPLE III 1-pr0pyl-2,4-dinitropyrrole A mixture of 38.6 grams (0.215 mole) of the sodium salt of 2,4-dinitropyrrole, 30 milliliters of propyl bromide and 30 milliliters of dimethylformamide is heated in a sealed tube at 135-140 C. for 3 /2 hours. After cooling, the tube is opened and the reaction mixture is worked up as described in Example I above. Distillation affords 24.7 grams of l-propyl-2,4-dinitropyrrole. This solid product is recrystallized from an ether-ligroin mixture to give 20.2 grams (47.2% yield) of pale yellow prisms, melting point 5657 C.

From the aqueous sodium carbon-ate solutions, a 35% recovery of 2,4-dinitropyrrole is effected an acidification with hydrochloric acid and extraction with ether.

EXAMPLE IV 1-(2,3-dihydr0xypr0pyl) -2,4-dinitr0pyrr0le A mixture of 5 grams (0.0275 mole) of the sodium salt chorohydrin, and 20 milliliters of dimethylformamide is heated under reflux for fifty minutes, cooled slightly, and then cencentrated under vacuum to remove the solvent. I'he viscous concentration residue is leached with three 300-milliliter portions of boiling ether, decanting each time from the insoluble residue. The combined ether solution is concentrated and the oily residue is distilled to afford the crude product as a viscous oil which boils at 200210 C., at 0.010.02 mm. It solidifies on standing and is recrystallized from ethyl acetate to give 2.4 grams (37.8% yield) of 1-(2,3-dihydroxypropyl) 2,4- dinitropyrrole, small cream prisms of melting point 111- 112 C.

This procedure is generally suitable for the preparation of water soluble 1-substituted-2,4-dinitropyrroles because it avoids the loss of these in aqueous wash solutions. It has been applied to the synthesis of the corresponding l-(fi-hydroxyethyl) and l-( -hydroxypropyl) analogs.

EXAMPLE V 1-(;3,'y-diacet0xypr0pyl)-2,4-dinitropyrr0le A mixture of 7.3 grams of l-(dy-dihydroxypropyl)-2,4- dinitropyrrole (Example IV) and 60 milliliters of acetic anhydride is treated under reflux for ninety minutes, cooled slightly, and then concentrated under vacuum to remove the excess of acetic anhydride. The syrupy residue is dissolved in 20 milliliters of boiling ethyl acetate and the solution is stored at to crystallize 8.4 grams of pale yellow granules which melt at 109-110". A second crop of 1.0 gram is isolated by concentration of the mother liquor. The total of 9.4 grams represents a yield of 94%.

EXAMPLE VI 1-carb0xymethyl-Z,4-dinitr0pyrr0le To a cold C.) solution of 5.2 grams (0.0214 mole) of 1-carbethoxymethyl-2,4-dinitropyrrole (prepared according to the process of Example II, from ethyl chloroacetate) in 150 milliliters of methanol is added 0.855 gram (0.0214 mole) of sodium hydroxide in milliliters of water. To the resulting paste is added 200 milliliters of water and this mixture is warmed at 45 C. for 10 minutes. After it has been cooled to 0 0., this solution is acidified with hydrochloric acid and the precipitated carboxylic acid is filtered off, washed on the filter with cold water, and dried in air. The 1-carboxymethyl-2,4-dinitropyrrole thus isolated, in almost quantitative yield, is hydrated. It melts at 205-207" C., after slow loss of water of hydration on the melting point stage.

Calcd. for C I-I O N C, 33.50; H, 2.34. Found: C, 33.63; H, 2.60.

EXAMPLE VII 1 (4 methylpiperazinocarbonylmethyl) 2,4- dim'tropyrrole A mixture of 10 grams of 1-carboxymethyl-Z,4-dinitropyrrole (Example VI) and 75 milliliters of thionyl chloride is boiled under reflux fortwo hours and then concentrated under vacuum at 60 C. The dark oily residue is dissolved in 50 milliliters of toluene and reconcentrated under vacuum at 60 C. The residue is dissolved in 100 milliliters of methylene chloride and this solution is stirred at 0 while a solution of 5 grams of N-methyL piperazine in 40 milliliters of methylene chloride is added over ten minutes. After bein allowed to stand at 25 for fifteen hours, the reaction mixture is shaken with 20 milliliters of 10% aqueous sodium carbonate and the methylene chloride layer is separated and dried with magnesium sulfate and then concentrated to a solid residue. This is recrystallized from ethyl acetate to afford 8.4 grams (61%) of 1-(4-methylpiperazino-carbonylmethyl)- 2,4-dinitropyrrole as bulf flakes which melt at 120-121". EXAMPLE VIII 1 (N -pyrrolidocarbonylmethyl -2,4 -dini trop yrrole A mixture of 10 grams of the sodium salt of 2.4-dinitropyrrole, 9.4 grams of N-chloroacetyl-pyrrolidine, and 40 milliliters of dimethylformamide is boiled under refiux for fifteen minutes and then poured into 600 milliliters of cold 5% aqueous potassium' carbonate. The insoluble solid is filtered off, dried in air and deeolorized with char- 1 The clear filtrate is evapr orated to dryness and the solid residue is recrystallized from ethyl acetate to afford 12 grams of 1-(1? pa e coal in an acetone solution.

pyrrolidinocarbonylmethyl)-2,4-dinitropyrrole as yellow prisms which melt at 168-159".

EXAMPLE IX (fi-bromoethyl)-2-mthyl-5-nitroimidazole Fifty milliliters of thionyl bromide is stirred vigorously and maintained at 50-60 while a total of 47.5 g. of l-(fi hydroxyethyl)-2-methyl-5-nitroimidazole is added .over a period of fifteen minutes. The reaction mixture is thenmaintained at 80-85" for fifteen minutes, cooled slightly,

and poured onto ice, the last of the contents being rinsed from the reaction vessel with methylene chloride and a little water. Three hundred milliliters of methylene chlo- EXAMPLE X 1-[ (2,4-dinitr0pyrryl-1 ethyl] -2-methyl-5 nitroimidazole A mixture of 17 grams of the potassium salt of 2,4- dinitropyrrole, 20 grams of l-(fi-bromoethyl)-2-methyl-5- nitroirnidazole, and 70 milliliters of dimethylformamide is stirred and boiled under reflux for fifteen minutes and then poured into one liter of cold 5% aqueous potassium carbonate. The insoluble solid is filtered ofi, dried in air and decolorized with charcoal in an acetone solution. The pale orange acetone filtrate (about 600 milliliters in volume) is boiled down to a volume of milliliters and chilled at 0 to effect crystallization of 16.4 grams of l-[2,4-dinitropyrryl-l)ethyl] 2 methyl-S-nitroimidazole, yellow granules which melt at 186-187".

A second crop of 2.1 grams is obtained on further concentration and chilling of the mother liquor, bringing the total to 18.5 grams, a yield of 70%.

The experimental details and physical contents of other compounds of the present invention prepared according to the methods illustrated in Examples I through X above, appear in Tables I, II and III.: The constants of com pounds not listed in Table III may be found in the detail d Examples I through X.

Reaction conditions (in Yield, percent dimethylformamide) TABLE III.Gontinued Analytical It B. P.C./mm. MP.

Calcd. Found dfiiciig CHZC ON lie-146"." 42.25 0; 4.26 41.91 C; 4,15 H.

CHCH:

cmooN N-CH| 12012l- 44.44o;5.0911..-- 44.42 o;s.-1e H CHgCHg CHgCON N-COCH; 215-21s- 44.31 0; 4.65 11.--. 44.09 0; 4,68 H.

CHrC I CHCHPN N 186187--. 38.71 C; 3.25H 39.09 0; 3.1911.

The in vitro trichomonadicidal activity of the compounds of the present invention may be demonstrated by a series of tests which establishes the minimal inhibitory concentration of these compounds. Minimal inhibitory concentration, as used above, is defined as the mininial concentration of a trichomonadicidal compound capable of preventing the growth of and killing Trichomonas foetus organisms introduced into a culture medium, capable alone of supporting a vigorous growth of the organisms and containing the trichomonadicidal compound to be tested. The culture medium used in the tests is described in a publication of Kupferberg, Johnson and Sprince, Proceedings of the Society for Experimental Biology and Medicine, volume 67, pages 304408, 1948.

In making the tests to determine minimal inhibitory concentrations, 0.05 milliliter of a 48-hour culture of T richomonas foetus is placed in a series of tubes containing milliliters of the culture medium and increasing amounts of the compound to be tested. The inoculated culture medium is then incubated at 37 C. for nine days and examined under magnification after two, five, seven, and nine days. The minimal inhibitory concentration of the compound tested is that concentration in the tube in which no viable organisms are present at the ninth day examination. If there are no viable organisms present on examination at a time less than nine days, the concentration of compound is greater than minimal and if there are viable organisms present at the ninth day examination,

the concentration is less than minimal. The results of these tests appear in column 1 of Table IV, wherein the concentration of the 1-snbstituted-ZA-dinitropyrroles is expressed in parts per million.

The toxicity of the compounds of the present invention are determined by oral administration to mice and may be determined from the in vivo data appearing in Table IV. In column 2, under the heading LD is indicated the quantity (in milligrams per kilogram of body weight) which is fatal to 50% of the mice tested. The amount (in milligrams per kilogram of body weight) required to cure 50% of the test animals injected with a lethal dose of Trichomonas foetus is indicated in column 3, under the heading PD In this column, N.P" indicates no protection. Column 4 of Table IV reports the therapeutic index of these compounds. A therapeutic index of at least 10 is preferred by pharmacologists as providing a suflicient margin of safety between the LD and the PD5Q.

TABLE IV.TRICHOMONADICIDAL ACTIVITY OF l-SUBSTITUTED'2,4

DINITROPYRROLES In vitro In vivo R activity,

p.p.m.

LDm PD) T.I.

n-C3 7 100 900 30 30. 0 n-CsHo 100.0 1, 200 53 23.0 iso C4Hu 10.0 1, 000 84 12. 0 sec-(hHn 10.0 1, 2004, 250 45 33. o n-CsHn 20. 0 1 500 50 30 iso-CsHn 100 1,100 5. 3 200. 0 sec-05H 20.0 2, 150 100 21. 5 n-CaHra 10. 0 2, 150 140-150 14. 3 cmomcHmr '10. 0 1, 4 120 12. 0- CHzCHzCHzCHzBl 0. 4 1, 000-2, 000 -100 10-20 CH2COCH3 10.0 1 000 87. 5 27. 0 CHZCH OH CHzOH 2. 0 1, 050 15-20 52. 0

O C O CH:

CHgCHCH2OCOCH3 10.0 1 900 25-30 76 OHgC H;

CHzC O-N /0 1.0 1, 500 5-7 200 OHQCH,

GET-CH3 CH C O-N N-CH; 10. 0 1, 050 10.5

TABLE iV.C'ontimred In vitro In vivo R activity,

p.p.m.

LD5u PDsc Tl.

C HzcHg CHgCON NCOCH3 10. 1, 050 67-135 10.

CH CHg CHKCHFN N (Inactive) 3, 000 10. 5 300 In employing the triohomonadicides of the present invention .for the treatment of Trichomonas foetus, one or more of the active agents are uniformly distributed in a suitable chemotherapeutic vehicle that is chemically compatible with the particular trichomonadicide selected, noninhibiting with respect to the action of the effective agent upon T richomonas foetus .and essentially non-injurious to the vaginal mucosa under the conditions of use. The vehicle is preferably of a liquid or semi-liquid type. Furthermore, since the final preparation should be readily miscible with vaginal fluids, the vehicles, whether hydrous or anhydrous, are preferably Water-miscible or water-dispersible. The compositions of this invention may be in the form of suppositories, if desired.

The foregoing criteria for a vehicle in which the compounds of the present invention are incorporated may be met by a large number of semi-liquid chemotherapeutic vehicles that are well known in the art. Thus, for example, the vehicle may comprise semi-liquids that are colloidal in nature, especially those that are viscous and/or mucilaginous in character. Such vehicles are particularly suitable for use in topical treatment of Trichomonas foetus because of their inherent gelatinous and miscible nature which affords prolonged contact between the l-substituted-2,4-dinitropyrrole and the infecting organism.

In order to disclose more clearly the manner of formulating the compounds of the present invention to topical application, several specific examples will hereinafter be described in considerable detail.

EXAMPLE XI Deionized water 75.80

Sodium carboxymethylcellulose 3.00

Polyethyleneglycol (molecular weight approximately 4000) 15.00

Propylene glycol 5.00 Para-hydroxyl-benzoic acid methyl ester 0.20 1-Methyl-2,4-dinitropyrrole 1.00

EXAMPLE XIII Deionized Water 90.30 Methyl cellulose 3.50 Glycerin 5.00 Para-hydroxy-benzoic acid methyl ester 0.20 1-methyl-2,4-dinitropyrrole 1.00

EXAMPLE XII Deionized Water 80.00

Pectin 8.00 Propylene glycol 10.00

Para-hydroxy-benzoic acid methyl ester 0.10 Para-hydroxy-benzoic acid propyl ester 0.10 l-(a-hydroxybutyl)-2,4-dinitropyrrole 1.00

The trichomonodicidal formulations of Examples XI through XIII are prepared according to the following general procedure in which two initial solutions are mixed to make the formulation, all the parts being given by weight. To prepare Solution A, dissolve the para-hydroxy-benzoic acid methyl ester in about two-thirds of the hot deionized Water, cool to about F., and, while stirring, add the gel-forming ingredient and glycerine or propylene glycol. To prepare Solution B, add the trichomonadicidal agent to the remainder of the deionized water, and adjust the pH to the desired value. The formulation is prepared by adding Solution B to Solution A in a slow stream with good stirring; stirring is continued for at least one hour.

Certain compounds of the present invention have also been found to be effective against enterohepatitis (blackhead) when administered by admixture, suspension, or dispersion in the food and/or drink normally partaken by turkeys, such as grain, mash, scratch, Water or other liquids.

The general range of concentration of the l-substituted- 2,4-dinitropyrrole in the total substance is from about 0.05% or less to about 1%. The optimal concentration for effective therapy is in the range from about 0.05% to about 0.2% of the total food or drinking water. With these optimal concentrations, the daily drug intake for infected birds varies from about 20 milligrams of drug per kilogram of body Weight to about 400 milligrams of drug per kilgram of body weight. In general, the precise dosage depends on the particular compounds and the severity of the infection. Many of the compounds of the present invention may be administered in the concentrations indicated above with little or no toxic effect.

Various changes and modifications of the invention may be made and to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claims.

What is claimed is:

A compound of the formula OzN-n No references cited.

HENRY R. IILES, Acting Primary Examiner. 

